Metabolic Blockade-Based Genome Mining of Sea Anemone-Associated Streptomyces sp. S1502 Identifies Atypical Angucyclines WS-5995 A–E: Isolation, Identification, Biosynthetic Investigation, and Bioactivities

Marine symbiotic and epiphyte microorganisms are sources of bioactive or structurally novel natural products. Metabolic blockade-based genome mining has been proven to be an effective strategy to accelerate the discovery of natural products from both terrestrial and marine microorganisms. Here, the metabolic blockade-based genome mining strategy was applied to the discovery of other metabolites in a sea anemone-associated Streptomyces sp. S1502. We constructed a mutant Streptomyces sp. S1502/Δstp1 that switched to producing the atypical angucyclines WS-5995 A–E, among which WS-5995 E is a new compound. A biosynthetic gene cluster (wsm) of the angucyclines was identified through gene knock-out and heterologous expression studies. The biosynthetic pathways of WS-5995 A–E were proposed, the roles of some tailoring and regulatory genes were investigated, and the biological activities of WS-5995 A–E were evaluated. WS-5995 A has significant anti-Eimeria tenell activity with an IC50 value of 2.21 μM. The production of antibacterial streptopyrroles and anticoccidial WS-5995 A–E may play a protective role in the mutual relationship between Streptomyces sp. S1502 and its host.


Introduction
Actinobacteria associated with plants or animals in marine environment produce various specialized metabolites (SMs) to compete with other microbes and adapt to the habitat [1,2], making them sources that human beings can use for clinical treat and agricultural production.Generally, the genome of an Actinobacteria usually contains at least 20 biosynthetic gene clusters (BGCs) that are responsible for the production of SMs; however, only limited amounts of BGCs are expressed under laboratorial culturing conditions, hindering the potential of further exploiting "talented strains".To block this limitation, manipulating the genome is a proven approach to activate lowly expressed or unexpressed BGCs by means of promoter refactoring [3], heterologous expression [4], and the overexpression of specific gene(s) such as regulatory or transport genes [5].Knocking out the core gene(s) related to identify known metabolites in a strain is a simple way of manipulating hindering the potential of further exploiting "talented strains".To block this limitation, manipulating the genome is a proven approach to activate lowly expressed or unexpressed BGCs by means of promoter refactoring [3], heterologous expression [4], and the overexpression of specific gene(s) such as regulatory or transport genes [5].Knocking out the core gene(s) related to identify known metabolites in a strain is a simple way of manipulating the genome that can result in an alternative metabolic flow, which might contribute to the identification of different or new SMs.This strategy has been widely applied to many strains including fungi and bacteria [6][7][8].Since common and shared precursors are utilized to assemble the main skeletons of most natural products, for example, polyketides use acyl-CoA(s) and malonyl-CoA(s), peptides use amino acids and terpenoids use farnesyl diphosphate (FPP) as building blocks, once the production of main chemical compositions is blocked in a strain, precursors can be otherwise used to generate other molecules; thus, novel SMs may be discovered.
Atypical angucyclines are polyketides that possess an angular tetracyclic benz[a]anthracene scaffold [9], and in some cases have much more complex ring systems that are synthesized by type II polyketide synthase and various post-modification enzymes including a vital JadG-type oxygenase, which directly leads to C-C bond cleavage on ring B/C of angucyclines [10,11].Extensive studies have been carried out for the discovery and biosynthesis of atypical angucyclines due to their intriguing structures and diversified bioactivities, particularly on jadomycins, gilvocarcins, kinamycins, and fluostatins (Figure 1) [9].Studies on biosynthetic compounds such as FAD-dependent monooxygenases, oxidoreductases, or other tailoring enzymes in fluostatins biosynthesis [12][13][14] and methyltransferases in gilvocarcins biosynthesis [15] extended the chemical landscape of atypical angucyclines; these enzymes catalyze special reactions and may be applied in synthetic biology to generate useful compounds.Meanwhile, heterologous expression of jadomycin, fluostatin, and kinamycins has been successfully achieved in different Streptomyces hosts [16][17][18][19].On the basis of the knowledge of biosynthetic logic of atypical angucyclines, recently, using a global genome-mining strategy of type II PKS, Zhang's group reported a type II PKS-containing biosynthetic gene cluster wsd which did not match previously reported type II PKS gene clusters in the MIBiG database from a soil-derived strain of Streptomyces yanglinensis CGMCC 4.2023, and this led to the discovery of a new compound: WS-5995D [20].Within our ongoing efforts to discover potential novel secondary metabolites with significant bioactivities or intriguing structures from marine microbial origins using the aforementioned metabolic blockade-based genome mining strategy [7,8], this study focuses on the potential bioactive metabolites of a sea anemone-associated Streptomyces sp.S1502.Chemical investigation of Streptomyces sp.S1502 led to the identification of three anti-methicillin-resistant Staphylococcus aureus (MRSA) halogenated pyrroles (halopyrroles)/streptopyrroles (including a new one, unpublished) [21].The core gene coding for AMP-dependent ligase for the first biosynthetic step of streptopyrroles was then inframe deleted to construct the mutant Streptomyces sp.S1502/Δstp1.Here, we report (1) five aromatic polyketides belonging to the family of atypical angucyclines, including four known compounds, WS-5995 A-D, and a new one, which herein we term as WS-5995 E, Within our ongoing efforts to discover potential novel secondary metabolites with significant bioactivities or intriguing structures from marine microbial origins using the aforementioned metabolic blockade-based genome mining strategy [7,8]

Identification of Streptopyrroles from Streptomyces sp. S1502 and the Construction of a Streptopyrroles-Null Mutant
In the process of searching for "talented strains" from our in-house symbiotic or epiphytic actinomycetes library associated with marine samples collected from Shenzhen Daya Bay on May, 2021, a strain of Streptomyces sp.S1502 isolated from a sea anemone sample caught our eyes during a systematic antibacterial assay screening of nearly 50 Actinobacterial strains, particularly due to its property against methicillin-resistant Staphylococcus aureus (MRSA) (Figure 2A) as well as other Gram-positive bacteria.To identify the corresponding compounds responsible for the anti-MRSA activity, three dominant metabolites were isolated from 23 L scaled fermentation of Streptomyces sp.S1502 and were identified as streptopyrroles (Figure 2B,D) according to their HR-MS spectrum and 1D NMR spectra (unpublished data) as well as a comparison with published data.This strain was subsequently whole-genome sequenced in order to determine the BGC linked to streptopyrroles biosynthesis and mine its potential abilities to produce other metabolites.The phylogenetic position of strain S1502 was established using autoMLST [22], indicating that it is not related to any other type of strain (Figure S1).The whole genome (~9.3 Mb) of Streptomyces sp.S1502 revealed that this strain contains at least 28 putative BGCs for polyketides, non-ribosomal peptides, ribosomal peptides, and terpenoids biosynthesis (Table S4) based on a bioinformatic analysis using antiSMASH 7.0 software [23].
were isolated and identified through 23 L scaled fermentation of the Δstp1 mutant; (2) the identification of corresponding BGC wsm, proposed biosynthetic pathway and heterologous production in S. lividans SBT5 of WS-5995 B-E; (3) probable tailoring and regulatory genes involved in WS-5995 A-E biosynthesis; and (4) the cytotoxic and in vitro anticoccidial bioactivities of WS-5995 A-E.

Identification of Streptopyrroles from Streptomyces sp. S1502 and the Construction of a Streptopyrroles-Null Mutant
In the process of searching for "talented strains" from our in-house symbiotic or epiphytic actinomycetes library associated with marine samples collected from Shenzhen Daya Bay on May, 2021, a strain of Streptomyces sp.S1502 isolated from a sea anemone sample caught our eye during a systematic antibacterial assay screening of nearly 50 Actinobacterial strains, particularly due to its property against methicillin-resistant Staphylococcus aureus (MRSA) (Figure 2A) as well as other Gram-positive bacteria.To identify the corresponding compounds responsible for the anti-MRSA activity, three dominant metabolites were isolated from 23 L scaled fermentation of Streptomyces sp.S1502 and were identified as streptopyrroles (Figure 2B,D) according to their HR-MS spectrum and 1D NMR spectra (unpublished data) as well as a comparison with published data.This strain was subsequently whole-genome sequenced in order to determine the BGC linked to streptopyrroles biosynthesis and mine its potential abilities to produce other metabolites.The phylogenetic position of strain S1502 was established using autoMLST [22], indicating that it is not related to any other type of strain (Figure S1).The whole genome (~9.3 Mb) of Streptomyces sp.S1502 revealed that this strain contains at least 28 putative BGCs for polyketides, non-ribosomal peptides, ribosomal peptides, and terpenoids biosynthesis (Table S4) based on a bioinformatic analysis using antiSMASH 7.0 software [23].Streptopyrroles belong to the family of pyrrole-containing alkaloids, members of this family also contain pyrrolomycin [24], pyoluteorin [25], chlorizidin [26], tetrachlorizine [26], and armeniaspirols [27].The first shared step of all established biosynthetic pathways related to compounds in this family is the activation and ligation with a carrier protein of proline, which is catalyzed by an amino acid (proline) adenyltransferase such as Arm4 (in armeniaspirol biosynthesis), PrlK (in pyrrolomycin biosynthesis), Tcz16 (in tetrachlorizine biosynthesis), or Clz14 (in chlorizidine biosynthesis).
Knowing the biosynthetic signature(s) of pyrrole-containing alkaloids, a BGC (BGC11, here designated as stp) sharing 37% of its identity with the reported pyrrolomycin BGC (prl) containing type I polyketide synthases and other tailoring enzymes were found in the genome of Streptomyces sp.S1502 (Figure 2C).Inside the gene cluster of stp, stp1 which shares a strong similarity with arm4, prlK, tcz16, and clz14 was present along with two genes stp2 and stp3 encoding dehydrogenase and halogenase, respectively, catalyzing the following reduction and chlorination of pyrrole, which is coincident with the biosynthesis of the above compounds.Thus, this gene cluster was deduced to be responsible for the biosynthesis of streptopyrrole.To verify this hypothesis, Streptomyces sp.S1502/∆stp1 (Streptomyces sp.S1502S) was constructed through in-frame deletion (Figure S2).A comparison of the metabolic profiling of this mutant with that of in wild-type strain revealed that the production of the streptopyrroles was completely abolished, and significantly increased yields of a series of peaks showing similar UV absorption were observed (Figure 3A).These results confirmed that stp is linked to streptopyrroles biosynthesis and a metabolic blockade strategy can be applied to this strain to mine other lowly expressed compounds.
anemone host, Streptomyces sp.S1502, and the anti-MRSA bioactivity of extract of Streptomyces sp.S1502 fermented in RA medium.(B) HPLC analysis of extract of Streptomyces sp.S1502; peaks marked with * are streptopyrroles.(C) The biosynthetic gene cluster stp responsible for streptopyrroles biosynthesis; stp1 for in-frame deletion is located in the middle of the cluster.(D) Proposed biosynthetic pathway of streptopyrroles.
Knowing the biosynthetic signature(s) of pyrrole-containing alkaloids, a BGC (BGC11, here designated as stp) sharing 37% of its identity with the reported pyrrolomycin BGC (prl) containing type I polyketide synthases and other tailoring enzymes were found in the genome of Streptomyces sp.S1502 (Figure 2C).Inside the gene cluster of stp, stp1 which shares a strong similarity with arm4, prlK, tcz16, and clz14 was present along with two genes stp2 and stp3 encoding dehydrogenase and halogenase, respectively, catalyzing the following reduction and chlorination of pyrrole, which is coincident with the biosynthesis of the above compounds.Thus, this gene cluster was deduced to be responsible for the biosynthesis of streptopyrrole.To verify this hypothesis, Streptomyces sp.S1502/Δstp1 (Streptomyces sp.S1502S) was constructed through in-frame deletion (Figure S2).A comparison of the metabolic profiling of this mutant with that of in wild-type strain revealed that the production of the streptopyrroles was completely abolished, and significantly increased yields of a series of peaks showing similar UV absorption were observed (Figure 3A).These results confirmed that stp is linked to streptopyrroles biosynthesis and a metabolic blockade strategy can be applied to this strain to mine other lowly expressed compounds.

Isolation and Structural Elucidation of Compounds from Streptomyces sp. S1502/∆stp1 Mutant
The mutant Streptomyces sp.S1502/∆stp1 was fermented at a 23 L scale in RA medium added with HP-20 resins for 7 days.Later, the extracts from the culture broth, resins, and mycelia were combined and subjected to consecutive fractionation using repeated silica gel column chromatography (CC).Prominent peaks in the HPLC analysis were purified by reverse-phase HPLC to produce compounds 1-5.
Compound 1 was obtained as a yellow solid.High-resolution electrospray ionization mass analysis afforded a [M + H] + ion at m/z 383.1122 (cald for 383.1125, Figure S3), suggesting that the molecular formula of 1 can be deduced to be that of C 21 H 18 O 7 and 13 degrees of unsaturation are required.The 1 H and 13 C NMR data (Table 1) showed a signal distribution typical for a tetracyclic benz[a]anthracene framework.The 1 H NMR spectrum (Figure S4) displayed signals for five aromatic (δ H 7.69, 7.64, 7.53, 7.27, and 6.99) and two methyl (δ H 3.77 and 2.44) signals.The combined analysis of 13 C NMR (Figure S5) and HMBC spectra (Figure S6) suggested the presence of 21 carbons including three carbonyls (δ C 185.0, 182.9, and 166.5), 12 aromatic (δ C 166. Compounds 2-5 were determined to be the known compounds WS-5995A, WS-5995B, WS-5995C, and WS-5995D, respectively, by analysis of their 1 H and 13 C NMR and HR-ESI-MS data (Figures S9-S20) [20,28,29].Genome sequence analysis of Streptomyces sp.S1502 revealed a contiguous region spanning ~30 kb containing 29 open reading frames (ORFs) that displayed a relatively high similarity and similar organization to the previously reported BGC (Figure 4A, wsd; MIBiG ID2712) [20].Remarkably, a homolog for almost every gene in the wsd gene cluster can be found in the wsm gene cluster in Streptomyces sp.S1502 strain, and every homologue in the WS-5595 BGC (wsd) shares a 58-90% amino acid sequence similarity and 46-81% of its identity with its corresponding gene associated with compounds 1-5 biosynthesis (Table S5).We noticed that some Streptomyces from different origins in public database also harbor this gene cluster with exactly the same gene composition or a highly similar organization (Figure S21), which indicates they are latent producers of the WS-5995 series of compounds.Compounds 2-5 were determined to be the known compounds WS-5995A, WS-5995B, WS-5995C, and WS-5995D, respectively, by analysis of their 1 H and 13 C NMR and HRESIMS data (Figures S9-S20) [20,28,29].

Characterization and Heterologous Expression of the wsm Biosynthetic Gene Cluster and Preliminary Investigation into the Genes Involved in Biosynthetic Pathways of Compounds 1-5
Genome sequence analysis of Streptomyces sp.S1502 revealed a contiguous region spanning ~30 kb containing 29 open reading frames (ORFs) that displayed a relatively high similarity and similar organization to the previously reported BGC (Figure 4A, wsd; MIBiG ID2712) [20].Remarkably, a homolog for almost every gene in the wsd gene cluster can be found in the wsm gene cluster in Streptomyces sp.S1502 strain, and every homologue in the WS-5595 BGC (wsd) shares a 58-90% amino acid sequence similarity and 46-81% of its identity with its corresponding gene associated with compounds 1-5 biosynthesis (Table S5).We noticed that some Streptomyces from different origins in public database also harbor this gene cluster with exactly the same gene composition or a highly similar organization (Figure S21), which indicates they are latent producers of the WS-5995 series of compounds.To verify whether the wsm gene cluster is responsible for WS-5995 A-E production, the ΔwsmA in-frame deletion mutant was constructed based on the Δstp1 mutant (Figure S22).HPLC analysis of the double mutant reveals the abolishment of WS-5995 A-E, proving that wsm is related to WS-5995 A-E production (Figure 4B).Some biosynthetic gene clusters of atypical angucyclines have been successfully heterologously expressed in different model microorganisms, thus extending the chemical diversity of this family of compounds and enabling the confirmation of the biosynthetic gene clusters.For example, an environmental DNA fragment containing type II PKS was heterologously expressed in To verify whether the wsm gene cluster is responsible for WS-5995 A-E production, the ∆wsmA in-frame deletion mutant was constructed based on the ∆stp1 mutant (Figure S22).HPLC analysis of the double mutant reveals the abolishment of WS-5995 A-E, proving that wsm is related to WS-5995 A-E production (Figure 4B).Some biosynthetic gene clusters of atypical angucyclines have been successfully heterologously expressed in different model microorganisms, thus extending the chemical diversity of this family of compounds and enabling the confirmation of the biosynthetic gene clusters.For example, an environmental DNA fragment containing type II PKS was heterologously expressed in Streptomyces albus J1074, resulting in the isolation of fluostatins [16].Coincidentally, fls from the marinederived fluostatins-producing strain Micromonospora rosaria SCSIO N160 was introduced into Streptomyces coelicolor YF11, leading to the production of a new dimer fluostatin, difluostatin A [18].Also, the gene cluster alp identified from S. galtieri Sgt26 was transferred into the heterologous host S. albus J1074 and the recombinant strain produced kinamycins [19].
To further link the relationship between WS-5995 A-E production and the proposed wsm gene cluster, heterologous expression of the wsm cluster in Streptomyces lividans SBT5 [30] and Streptomyces atratus ZH16NSEP (our genetic-engineered marine-derived chassis cell for a scaled heterologous expression platform of marine microbial bioactive metabolites) [31] was performed.A bacterial artificial cosmid library of the S1502 strain was constructed.We screened a recombinant plasmid pBAC/1-9A carrying the intact wsm BGC from the bacterial artificial chromosome (BAC) library of Streptomyces sp.S1502/∆stp1.The plasmid pBAC/1-9A was then introduced into the two heterologous hosts, respectively, via triparental intergeneric conjugation, generating Streptomyces lividans SBT5: pBAC/1-9A and Streptomyces atratus ZH16NSEP: pBAC/1-9A.HPLC analysis of the heterologous strain fermentation extract revealed that the wsm cluster could be solely successfully expressed in S. lividans SBT5 and could stably produce WS-5995 B-E, and while WS-5995 A was not detected, the yields of WS-5995 B-E were relatively lower compared to the wild-type producer (Figure 4B).
According to bioinformatic analysis of each gene in wsm BGC, the biosynthesis of WS-5995 A-E is proposed (Figure 4C).A common biosynthetic pathway of aldehyde/acid intermediate of atypical angucyclines is proposed as follows: WsmA, WsmB, and WsmC utilize one acetyl-CoA unit and nine malonyl-CoA units to form the initial 20-carbon polyketide chain, which is reduced at position C-9 by WsmD and cyclized by WsmE and WsmU, before further functioned by WsmF and WsmT; then, the common intermediate dehydrorabelomycin is formed.Subsequently, the methyltransferase WsmH is proposed to hydroxylate the -OH group of ring D prior to the GilOII/JadG homolog WsmI (Figure S23) catalyzing the C-C bond cleavage of ring C to form the aldehyde/acid intermediate, as once the carboxyl group is formed, it might react with the hydroxylation group to form an ester product, similar to gilvocarcins biosynthesis [11], while such products were not isolated from Streptomyces sp.S1502/∆stp1 mutant.Decarboxylation and oxidation of the aldehyde group of the aldehyde/acid intermediate leads to the formation of WS-5995 B, and hydroxylation on the position of decarboxylation forms WS-5995 C, which are probably catalyzed by WsmO/P/Q/R; the four protein homologs are also present in wsd gene cluster.Last, intramolecular esterification of WS-5995 C and intermolecular esterification of WS-5995 C and methanol/ethanol were carried out to form WS-5995 A and WS-5995 D/E.
The results of the deletion of the KS-coding gene wsmA and heterologous expression of wsm gene cluster unambiguously confirmed the correlation between WS-5995 A-E production and the wsm gene cluster; we next sought to identify putative genes involved in the final steps of WS-5995 A-E biosynthesis.A question can be raised about whether WS-5995E (1) is an artificial product, as during the extraction process, the reason that the ethanol was used, pure WS-5995 C (4) was dissolved in methanol and ethanol for 72 h, respectively, we did not detect the production of WS-5995A (2), D (5), or E (1) (Figure S24).To further verify this, Streptomyces sp.S1502 was re-fermented in RA medium without the addition of resins, and it was extracted with butanone.The HPLC analysis results showed that WS-5995 A-E were produced too.These two observations indicate that WS-5995A, D, and E are not spontaneous products, indicating that the formation of an ester bond is more likely to be an enzymatic process.
To identify the function of tailoring and regulatory genes involved in WS-5995 A-E biosynthesis, the wsm gene cluster and wsd gene cluster were re-examined and compared using a clinker [32] (Figure S25).Some genes are relatively special in wsm/wsd gene clusters compared to other atypical angucycline biosynthetic gene clusters, including wsmO, P, Q, R, X, Y, and O 3 (in wsm).These genes might be involved in the tailoring steps in the creation of WS-5995 A-E, and therefore, were individually deleted (Figure S26).The corresponding mutants were fermented; the ∆wsmO and ∆wsmO 3 mutants retained the production of WS-5995 A-E, the former also produced two new products (a and b), while the latter had a lower yields of the five compounds; the ∆wsmP, ∆wsmX and ∆wsmY mutants abolished the production of WS-5995 A-E, and the ∆wsmR and ∆wsmQ mutants only produced WS-5995 C (4) (Figure 5i-viii).According to the HPLC analysis of the above mutants, all seven of the genes have been preliminarily deduced to be involved in WS-5995 biosynthesis; with the result that the heterologous expression of wsm gene cluster did not produce WS-5995 A (2), indicated that the enzyme catalyzing WS-5995 C (4) to WS-5995 A (2) might be located outside of the gene cluster.
5995 C (4) (Figure 5i-viii).According to the HPLC analysis of the above mutants, all seven of the genes have been preliminarily deduced to be involved in WS-5995 biosynthesis; wsmO3 may have a homolog in the Streptomyces sp.S1502 genome that has a complemental function to WsmO3; WsmP, WsmX, and WsmY are involved the tailoring steps of the aldehyde/acid intermediate; WsmR and WsmQ may participate in the transformation of WS-5995 C (4) to WS-5995 A (2); and WsmO might catalyze an unknown reaction.Combining these results with the result that the heterologous expression of wsm gene cluster did not produce WS-5995 A (2), indicated that the enzyme catalyzing WS-5995 C (4) to WS-5995 A (2) might be located outside of the gene cluster.The role of the four regulatory genes wsmR 1 -R 4 in WS-5995 A-E biosynthesis was also characterized to provide a mutant that might have higher yields of WS-5995 A-E.The four genes were individually deleted (Figure S26) and the mutants were fermented.As seen from the HPLC analysis, wsmR 1 , R 2 , and R 4 have been preliminarily deduced to have a positive role, while it appears that wsmR 3 has a negative role in WS-5995 A-E biosynthesis (Figure 5ix-xii).Thus, the ∆wsmR 3 mutant is a relatively high-producing strain of WS-5995 A-E.

Bioactivities of WS-5995 A-E
Four human cancer cell lines (MCF-7, BT-549, RBE and A549) and two normal human cell lines (NCM-46 and Huvec) were used to test the cytotoxicity of compounds 1-5 (Table 2).Compounds 1, 4, and 5 did not exhibit cytotoxic effects against the abovementioned cell lines, while both compounds 2 and 3 showed slight cytotoxicity against the RBE, NCM-460, and Huvec cell lines, with IC 50 values of 21.39, 40.84, and 31.24µM for compound 2 and 16.79, 18.47, and 7.67 µM for compound 3, respectively.In addition, compound 3 was also found to show cytotoxicity against MCF-7 and BT-549 cell lines, with IC 50 values of 19.60 and 5.97 µM, respectively.From the structure and the activities, we proposed that the ester bond might be important to cytotoxic activity, and hydroxylation at C-3 can lead to the removal of cytotoxic activity.A previous study established that WS-5995 A (2) and B (3) showed excellent protective activity against Eimeria tenella infections in animal experiments [29].However, the in vitro anticoccidial activity is still lacking experimental data.Compounds 1-5 were evaluated for their in vitro anti-E.tenella bioactivity.WS-5995 A (2) had clear anti-E.tenella activity with an IC 50 value of 2.21 µM, and while compound 4 is not active, compounds 1 and 5 only exhibited activity at high concentrations.These results coincide with the animal experiments reported previously.The in vitro anticoccidial activity showed that the lactone bond of WS-5995 A is vital to this bioactivity.

General Experimental Procedures
DNA isolation and manipulation were carried out following standard procedures for E. coli and Streptomyces.Reagents for polymerase chain reactions (PCRs) were purchased from Takara Co. (Dalian, China) and Trans Gene Co. (Beijing, China).The plasmid extraction kit and gel extraction kit were purchased from Omega (Beijing, China).The media used for fermentation were purchased from Guangdong Huankai Microbial Technology Co., Ltd.(Guangzhou, China).Luria-Bertani (LB) broth used for E. coli cultivation was purchased from Oxoid Co., Ltd.(Hants, UK).Organic solvents for compounds isolation and purification were bought from Guangzhou Chemical Regent Factory (Guangzhou, China) or J&K Scientific Co., Ltd.(Beijing, China).All chemicals and solvents were of analytical or chromatographic grade.All primers and reagents used in this work were purchased from Sangon Bio-Pharm Technology Co., Ltd.(Shanghai, China).

Strains, Plasmids, and Culture Conditions
The strain S1502 was isolated from a sea anemone sample collected from Shenzhen Daya Bay in May 2021, and it was identified as Streptomyces sp.based on its morphological characteristics as well as phylogenetic analysis of the 16S rRNA gene sequence.

Genome Sequencing and Bioinformatic Analysis
The secondary metabolite BGCs of Streptomyces sp.S1502 were identified and analyzed using the online software antiSMASH version 7.1.0[23].The orf assignments and their proposed functions were analyzed using the online FramePlot 4.0beta (http://nocardia.nih.go.jp/fp4/, accessed on 7 June 2022).Functional prediction and domain annotation of orfs were accomplished using BLAST software (http://blast.ncbi.nlm.nih.gov/,accessed on 7 June 2022).Sequence alignments were achieved using MEGA 7.0 and Clustal X.

Construction of the ∆stp1 and Mutants Based on the ∆stp1
λ-RED mediated PCR-targeting method was applied in this study to inactivate the targeted genes [31,33].First, a 1384 bp apramycin resistance gene as a PCR template was obtained through digestion of plasmid pIJ773 by HindIII and EcoRI.Then, each of the apramycin resistance gene cassettes (aac(3)IV-oriT) was amplified by PCR using the obtained template with designed primers listed in Table S2.Subsequently, the PCR-targeting and conjugation processes were carried out as described previously [31].The construction process of in-frame deletion strp1 was similar to our previous report and can simply be described as follows.
For stp1 in-frame deletion, the Supercos I vector-based cosmid 10-11F harboring the partial streptopyrrole cluster containing stp1 was used.The apramycin resistance gene cassette (aac(3)IV-oriT) was amplified by PCR using the obtained template (del-stp1-F and del-2970-R) with the designed primers listed in Table S2.The apramycin resistance cassette was introduced into E. coli BW25113/pIJ790/10-11F by chemical transformation to disrupt the homologous gene region to generate cosmid 10-11F-a.The mutated cosmid 10-11F-a was digested with SpeI and subsequently self-ligated to generate 10-11F-b which was verified with the primers listed in Table S2.Then, the kanamycin resistance gene of the cosmid 10-11F-b was replaced by the aac(3)IV-oriT cassette and transferred to non-methylating E. coli ET12567/pUZ8002, and then introduced into S. sp.S1502 by conjugation.Exconjugants were verified with the primers listed in Table S2 to generate single crossover mutants and then to generate double-crossover mutant indel-∆stp1 after two generations of relaxed cultivation.
The construction of the ∆wsmA mutant followed the same process of ∆stp1 mutant based on the ∆stp1 mutant.Other mutants were constructed by replacing the deleted fragments of cosmids 13-4D or 13-5D with the apramycin resistance gene cassette (aac(3)IV-oriT).

Heterologous Expression of the wsm Gene Cluster in S. lividans SBT5
The bacterial artificial chromosome (BAC) genomic library of Streptomyces sp.S1502 was constructed by Eight Star Biotech [34] (http://www.eightstarsbio.com,accessed on 1 January 2023) using the vector pMSBBAC2 and stored in E. coli DH10B.The BAC library was screened by PCR using the primers to afford the desired colonies pBAC1-9C/1-16A, which contain the intact wsm BGC.pBAC1-9C/1-16A was used for conjugation with S. lividans SBT5 and S. atratus SCSIO ZH16NSEP using the triparental conjugation methods and cultured on MS or ISP4 medium.The correctness of the conjugants was verified by PCR analysis.

Large-Scale Fermentations of Streptomyces sp. S1502 and ∆stp1 Mutant
A portion of mycelium and spores of the wild-type strain or the ∆stp1 mutant of Streptomyces sp.S1502 were seeded into 50 mL RA medium in a 250 mL flask and a 23 L scaled fermentation was carried out.After incubation in a shaker at 28 • C, 200 rpm for 48 h, 25 mL of the culture was transferred into a 1 L flask containing 200 mL RA medium and then cultured at 28 • C and 200 rpm for 7-8 d.The growth culture was centrifuged at 3900 g for 10 min to yield supernatant and mycelium cake.The supernatant, mycelium, and resins were independently extracted by twice the volume of butanone, acetone, and ethanol three times, respectively.The organic extracts were combined and concentrated to dryness, yielding a syrupy residue.

Cytotoxic Activities of Compounds 1-5
Compounds 1-5 were tested for their cytotoxic activities against different normal and tumor human cell lines, the MTT assay was used to assess cytotoxicity as described previously.Briefly, different tumor cells and normal cells were seeded onto 96-well plates at the appropriate density and allowed to attach overnight.Drugs at various concentrations were added to the culture medium and cells were cultured for another 68 h at 37 • C. MTT (5 mg/mL, 20 µL/well) was then added to the wells for additional 4 h, and the medium was discarded before 150 µL of DMSO was added to dissolve the MTT-formazan crystals.Absorbance was measured at 540 nm, with background subtraction at 670 nm using Model 550 Microplate Reader (Bio-Rad, Hercules, CA, USA).The Bliss method was used to calculate the half maximal (50%) inhibitory concentration (IC 50 ) values.Data represent the mean ± SD of at least three independent experiments.

In Vitro Anticoccidial Activity
The number of Eimeria tenella spores and the cells were seeded in a 24-well plate at a ratio of 1:1 and incubated for 6 h, and the medium was discarded and washed with PBS three times.Then, 500 µL of medium was added to each well, followed by the addition of the medium containing the compounds, the blank control group (no spores and no compounds) and positive control group (with spores but no compound) were set, and 10 µM of sulfaclozinium chloride sodium served as a control drug, and three replicates were set up.The culture remained in an incubator containing 5% CO 2 at 38 • C for 48 h.After 48 h, the medium was discarded and washed with PBS three times, and the total RNA of each well was extracted and reverse-transcribed into cDNA.qPCR was used to detect the effects of the compounds on the growth and development of E. tenella.

Discussion and Conclusions
In the long history of evolution, actinomycetes and their related marine or terrestrial animals might have had a mutual relationship, for the reason that they produce antibiotics to protect the hosts and utilize the excellent environment provided by the host, as can be seen from some other studies [2,35,36].To take advantage of this, natural products from these microorganisms have drawn much more attention as they seem to be more potent and can be developed into drugs.The marine symbiotic microorganisms harbor vast BGCs to help their host adapt to the extreme marine environment and have great potential to produce various novel natural products.However, only limited BGCs are active in one strain and many natural products are thus only rediscovered under conventional experimental cultivation conditions.With the rapid advancement of genome sequencing and the development of bioinformatics, numerous genome mining strategies have been applied to discover novel natural products, but many attempts have been in vain.It is worth mentioning that one of the genome mining strategies, metabolic blockade-based genome mining, is efficient and can be used to discover novel natural products [1,[6][7][8].
Atypical angucyclines are a type of natural product possessing an angular tetracyclic benz[a]anthracene scaffold [9] synthesized by type II polyketide synthase and various postmodification enzymes.The WS-5995 type of compounds belonging to atypical angucyclines were isolated from the Streptomyces sp.S1502 by metabolic blockade-based genome mining strategy in this study, and our further study revealed that many post-modification enzymes' genes are involved in the biosynthesis of WS-5995.The characterization of regulatory genes in wsm BGC led to the generation of a high titer production of the engineered WS-5995 strain, which will be helpful for the further study of WS-5995 compounds.In addition, the bioactivity test of these five compounds showed that WS-5995 A has significant anticoccidial activity and is non-toxic against the tested cell lines, which indicated that it has great potential to be developed into anticoccidial drugs.In addition, WS-5995 compounds exhibited broad-spectrum antibacterial and anticoccidial activities, respectively; in addition, WS-5995B was also reported to have antifungal activity [28], which led us to speculate here that this bacterium may use these compounds to protect the sea anemone host from infections from specific pathogenic bacteria/fungi and protozoan, explaining the plausible ecological functions of these compounds produced by this sea anemoneassociated bacterium.
In summary, five atypical angucyclines, WS-5995 A-E, were discovered through the metabolic blockade-based genome mining strategy from a sea anemone-associated Streptomyces sp.S1502 ∆stp1 mutant, among which WS-5995 E is a new compound.The BGCs (wsm) of WS-5995 A-E were identified by combining gene-knockout experiments and heterologous expression studies, and some tailoring and regulatory genes involved in WS-5995 A-E biosynthesis were also investigated.The gene knockout results indicated that WsmO 3 , O-R, X, and Y are involved in WS-5995 A-E biosynthesis, but their specific roles are yet to be confirmed; the regulatory gene wsmR3 had a negative effect on the production of WS-5995 A-E, and the deletion of this gene clearly increased the yield of WS-5995 A-E.The gene cassette wsmO-R can be regarded as a unique signature compared to other atypical angucyclines BGCs.
, this study focuses on the potential bioactive metabolites of a sea anemone-associated Streptomyces sp.S1502.Chemical investigation of Streptomyces sp.S1502 led to the identification of three anti-methicillin-resistant Staphylococcus aureus (MRSA) halogenated pyrroles (halopyrroles)/streptopyrroles (including a new one, unpublished) [21].The core gene coding for AMP-dependent ligase for the first biosynthetic step of streptopyrroles was then inframe deleted to construct the mutant Streptomyces sp.S1502/∆stp1.Here, we report (1) five aromatic polyketides belonging to the family of atypical angucyclines, including four known compounds, WS-5995 A-D, and a new one, which herein we term as WS-5995 E, were isolated and identified through 23 L scaled fermentation of the ∆stp1 mutant; (2) the identification of corresponding BGC wsm, proposed biosynthetic pathway and heterologous production in S. lividans SBT5 of WS-5995 B-E; (3) probable tailoring and regulatory genes involved in WS-5995 A-E biosynthesis; and (4) the cytotoxic and in vitro anticoccidial bioactivities of WS-5995 A-E.Mar.Drugs 2024, 22, 195 3 of 15

Figure 2 .
Figure 2. Streptopyrroles from Streptomyces sp.S1502 and their biosynthesis.(A) Images of the sea anemone host, Streptomyces sp.S1502, and the anti-MRSA bioactivity of extract of Streptomyces sp.S1502 fermented in RA medium.(B) HPLC analysis of extract of Streptomyces sp.S1502; peaks marked with * are streptopyrroles.(C) The biosynthetic gene cluster stp responsible for streptopyrroles biosynthesis; stp1 for in-frame deletion is located in the middle of the cluster.(D) Proposed biosynthetic pathway of streptopyrroles.
Mar. Drugs 2024, 22, 195 6 of 15 2.3.Characterization and Heterologous Expression of the wsm Biosynthetic Gene Cluster and Preliminary Investigation into the Genes Involved in Biosynthetic Pathways of Compounds 1-5

Figure 4 .
Figure 4. Proposed biosynthesis of WS-5995 A-E.(A) Comparison of wsm and wsd gene clusters and their gene organization.(B) Confirmation of wsm gene cluster by HPLC analysis.(C) Proposed biosynthetic pathway of WS-5995 A-E.

Figure 4 .
Figure 4. Proposed biosynthesis of WS-5995 A-E.(A) Comparison of wsm and wsd gene clusters and their gene organization.(B) Confirmation of wsm gene cluster by HPLC analysis.(C) Proposed biosynthetic pathway of WS-5995 A-E.
wsmO 3 may have a homolog in the Streptomyces sp.S1502 genome that has a complemental function to WsmO 3 ; WsmP, WsmX, and WsmY are involved the tailoring steps of the aldehyde/acid intermediate; WsmR and WsmQ may participate in the transformation of WS-5995 C (4) to WS-5995 A (2); and WsmO might catalyze an unknown reaction.Combining these results Mar.Drugs 2024, 22, 195 8 of 15